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Research & Scholarship

Current Research and Scholarly Interests

Our lab studies how the local tissue microenvironment contributes to immunity and immune regulation. We work at the intersection of immunology and structural biology.

Major areas of investigation include:

The Tissue Microenvironment and Immune Regulation in DiabetesWe are studying the regulatory mechanisms that turn on and off immune responses within inflamed tissues. In particular, we focus on interactions between hyaluronan (HA) , a prominent component of inflamed extracellular matrix, and regulatory T-cells in type 1 diabetes (T1D). Our data suggest that the size and amount of HA polymers within injured and healing tissues provide contextual cues that help govern local immune responses. In addition to elucidating the underlying mechanisms, we are working on novel therapeutic strategies that will prevent T1D by targeting the extracellular matrix in autoimmune insulitis.

The Immunology of Diabetic WoundsChronic wounds, like tissues under autoimmune attack, are associated with an inflamed extracellular matrix that contributes to immune dysregulation and chronic wounds. We use models of diabetic wound healing to study how the extracellular matrix governs wound healing and local immunity.

Microbial Biofilms and Diabetic Wound InfectionsThe virulence of the major human pathogen Pseudomonas aeruginosa is predicated on its ability to form biofilms. These are networks of host and microbial extracellular matrix that promote colonization, antibiotic resistance, and immune evasion. We are studying how polymers and bacterial factors within those biofilms suppress local immune responses in infected wounds. Further, we are developing innovative strategies to treat chronic infections by disrupting microbial biofilms.

Abstract

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Abstract

Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.

Abstract

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Abstract

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Abstract

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

Abstract

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.

Abstract

Group III Pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood.Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis.In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA dependent manner.Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.

Abstract

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Abstract

Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied.It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 β (IL-1β) in an NFkB dependent manner and blocking antibodies to IL-1β abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation.Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1β, our results suggest that therapeutic anti-IL-1β antibodies, currently tested in human clinical trials, may exert their beneficial effects on inflammation in post-myocardial infarction patients via effects on HAS3. in post-myocardial infarction patients who remain at high cardiovascular risk due to persistent elevated inflammatory biomarkers.

Abstract

Humans express an enzyme that degrades chitin, called chitotriosidase, despite the fact that we do not produce chitin. One possible explanation for this is that chitinase also degrades hyaluronan, a polysaccharide that is abundant in human tissues and shares structural attributes in common with chitinase. The objective of this study was to determine whether human chitotriosidase is capable of hydrolyzing hyaluronan. Hyaluronan of various sizes under a range of pH conditions displayed no degradation when incubated with various chitinases over a period of 5 days, while commercial hyaluronidase readily digested the hyaluronan. Under the same conditions, recombinant chitinase but not our negative control chitinase, was able to digest chitosan. We conclude that human chitinase does not digest hyaluronan. Because chitin is a prominent component of certain fungi and insects, it seems likely that human chitinase evolved for roles in host defense rather than serving to catabolize the endogenous polymer hyaluronan.

Abstract

We have identified a novel role for hyaluronan (HA), an extracellular matrix (ECM) polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes (T1D) of mice and humans is preceded by intra-islet accumulation of HA, a highly hygroscopic polymer. Using the DORmO double transgenic (DO11.10 x RIPmOVA) mouse model of T1D, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared to controls. Conversely, treatment with 4-methylumbelliferone (4-MU), a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Abstract

Tertiary lymphoid follicles (TLFs) can develop in the respiratory tract in response to infections or chronic inflammation. However, their functional relevance remains unclear as they are implicated in both protective and pathologic responses. In contrast to homeostatic conditions, external antigens and damage to the lung tissue may drive TLF formation in inflamed lungs, and once established, the presence of pulmonary TLFs may signal the progression of chronic lung disease. This novel concept will be discussed in light of recent work in chronic obstructive pulmonary disease (COPD) and how changes in the pulmonary microbiota may be drive and direct TLF formation and function. We will also discuss the cellularity of TLFs at the pulmonary mucosa, with emphasis on the potential roles of LTi's, B and T cell aggregates and examine the function of key chemokines and cytokines including CXCL13 and IL-17, in the formation and maintenance of pulmonary TLFs. This article is protected by copyright. All rights reserved.

Abstract

Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan synthesis in mouse models of cancer, autoimmunity, and a variety of other inflammatory disorders where hyaluronan (HA) has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration, and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after one week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a one-week loading period on the drug is required for most protocols. At steady state, over 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulfated metabolite, 4-methylumbelliferyl sulfate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0.65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice. This article is protected by copyright. All rights reserved.

Abstract

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Abstract

Significance: Fetal wounds heal with a regenerative phenotype that is indistinguishable from surrounding skin with restored skin integrity. Compared to this benchmark, all postnatal wound healing is impaired and characterized by scar formation. The biologic basis of the fetal regenerative phenotype can serve as a roadmap to recapitulating regenerative repair in adult wounds. Reduced leukocyte infiltration, likely mediated, in part, through changes in the chemokine milieu, is a fundamental feature of fetal wound healing. Recent Advances: The contributions of chemokines to wound healing are a topic of active investigation. Recent discoveries have opened the possibility of targeting chemokines therapeutically to treat disease processes and improve healing capability, including the possibility of achieving a scarless phenotype in postnatal wounds. Critical Issues: Successful wound healing is a complex process, in which there is a significant interplay between multiple cell types, signaling molecules, growth factors, and extracellular matrix. Chemokines play a crucial role in this interplay and have been shown to have different effects in various stages of the healing process. Understanding how these chemokines are locally produced and regulated during wound healing and how the chemokine milieu differs in fetal versus postnatal wounds may help us identify ways in which we can target chemokine pathways. Future Directions: Further studies on the role of chemokines and their role in the healing process will greatly advance the potential for using these molecules as therapeutic targets.

Abstract

The diabetic phenotype of wound healing is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Angiopoietin-1 (Ang-1) is a potent mobilizer of EPCs from the bone marrow (BM). A suggested mechanism for EPC mobilization from the BM is mediated by matrix metalloproteinase 9 (MMP-9) and stem cell factor (SCF). Taken together, we hypothesized that overexpression of Ang-1 in diabetic wounds will recruit EPCs and improve neovascularization and wound healing.An endothelial lineage BM-labeled murine model of diabetes was developed to track BM-derived EPCs. FVBN mice were lethally irradiated and then reconstituted with BM from syngeneic Tie2/LacZ donor mice. Diabetes was induced with streptozotocin. Dorsal wounds in BM-transplanted mice were treated with Ad-Ang-1, Ad-GFP, or phosphate-buffered saline. At day 7 after injury, wounds were harvested and analyzed. A similar experiment was conducted in EPC mobilization deficient MMP-9 -/- mice to determine whether the effects of Ang-1 were EPC-dependent.Overexpression of Ang-1 resulted in greatly improved re-epithelialization, neovascularization, and EPC recruitment in diabetic BM-transplanted wounds at day 7. Ang-1 treatment resulted in increased serum levels of proMMP-9 and SCF but had no effect on vascular endothelial growth factor levels. According to our FACS results, peripheral blood EPC (CD34(+)/Cd133(+)/Flk1(+)) counts at day 3 after wounding showed impaired EPC mobilization in MMP-9 -/- mice compared with those of wild-type controls. EPC mobilization was rescued by SCF administration, validating this model for EPC-mobilization-deficient mechanistic studies. In MMP-9 -/- mice, Ad-Ang-1 accelerated re-epithelialization in a similar manner, but had no effect on neovascularization.Our results show that Ang-1 administration results in improved neovascularization which is dependent on EPC recruitment and has direct effects on wound re-epithelialization. These data may represent a novel strategy to correct the phenotype of impaired diabetic neovascularization and may improve diabetic wound healing.

Abstract

Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process.To evaluate the differences in functional profile, we compared metabolism (MTS assay), apoptosis (caspase-3 staining), migration (scratch wound assay) and invasion (transwell assay) between C57Bl/6J murine fetal (E14.5) and adult (8 weeks) fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner.Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.

Abstract

Cyclosporine A (CSA) is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg) through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA), promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA.

Abstract

Hyaluronan is an extracellular matrix glycosaminoglycan that is present in pancreatic islets but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and non-diabetic organ donors differ in the amount and distribution of hyaluronan and hyaluronan binding proteins (hyaladherins) such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor-stimulated gene-6 (TSG-6). Hyaluronan was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, hyaluronan was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in hyaluronan-rich areas of islets and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of less than 10 years. Furthermore, hyaluronan and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D compared to controls. Our observations highlight potential roles for hyaluronan and hyaladherins in the pathogenesis of diabetes.

Abstract

Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing.Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing.Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls.The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.

Abstract

The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates "tissue integrity signals" that promote resolution of local immune responses.

Abstract

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.

Abstract

Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses.To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study.Subjects received 4 weekly doses of rituximab (N = 57) or placebo (N = 30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology.EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p < 0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p < 0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic.Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses.

Abstract

Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

Abstract

Type 1 diabetes (T1D) is a disease that in most individuals results from autoimmune attack of a single tissue type, the pancreatic islet. A fundamental, unanswered question in T1D pathogenesis is how the islet tissue environment influences immune regulation. This crosstalk is likely to be communicated through the extracellular matrix (ECM). Here, we review what is known about the ECM in insulitis and examine how the tissue environment is synchronized with immune regulation. In particular, we focus on the role of hyaluronan (HA) and its interactions with Foxp3+ regulatory T-cells (Treg). We propose that HA is a "keystone molecule" in the inflammatory milieu and that HA, together with its associated binding proteins and receptors, is an appropriate point of entry for investigations into how ECM influences immune regulation in the islet.

Abstract

The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T-cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T-lymphocytes on ECM made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM, rich in versican and hyaluronan, to which T-cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T-lymphocytes. Using purified matrix components and solid state matrices of defined composition, we uncovered a role for versican in modulating hyaluronan-T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan-coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.

Abstract

We have developed a bioengineered implant (BI) to evaluate strategies to promote graft survival and function in models of islet transplantation in mice. The BI, sized for implantation within a fold of intestinal mesentery, consists of a disk-shaped, polyvinyl alcohol sponge infused with a type I collagen hydrogel that contains dispersed donor islets. To promote islet vascularization, the BI incorporates a spherical alginate hydrogel for sustained release of vascular endothelial growth factor (VEGF). BIs that contained 450-500 islets from syngeneic (C57Bl/6) donors and 20 ng of VEGF reversed streptozotocin (STZ)-induced diabetes in 100% of mice (8/8), whereas BIs that contained an equivalent number of islets, but which lacked VEGF, reversed STZ-induced diabetes in only 62.5% of mice (5/8). Between these "+VEGF" and "-VEGF" groups, the time to achieve normoglycemia (8-18 days after implantation) did not differ statistically; however, transitory, postoperative hypoglycemia was markedly reduced in the +VEGF group relative to the -VEGF group. Notably, none of the mice that achieved normoglycemia in these two groups required exogenous insulin therapy once the BIs began to fully regulate levels of blood glucose. Moreover, the transplanted mice responded to glucose challenge in a near-normal manner, as compared to the responses of healthy, nondiabetic (control) mice that had not received STZ. In future studies, the BIs described here will serve as platforms to evaluate the capability of immunomodulatory compounds, delivered locally within the BI, to prevent or reverse diabetes in the setting of autoimmune (type 1) diabetes.

Abstract

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.

Abstract

Glutamic acid decarboxylase (GAD) is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. In animal models of autoimmunity, treatment with a target antigen can modulate aggressive autoimmunity. We aimed to assess whether immunisation with GAD formulated with aluminum hydroxide (GAD-alum) would preserve insulin production in recent-onset type 1 diabetes.Patients aged 3-45 years who had been diagnosed with type 1 diabetes for less than 100 days were enrolled from 15 sites in the USA and Canada, and randomly assigned to receive one of three treatments: three injections of 20 μg GAD-alum, two injections of 20 μg GAD-alum and one of alum, or 3 injections of alum. Injections were given subcutaneously at baseline, 4 weeks later, and 8 weeks after the second injection. The randomisation sequence was computer generated at the TrialNet coordinating centre. Patients and study personnel were masked to treatment assignment. The primary outcome was the baseline-adjusted geometric mean area under the curve (AUC) of serum C-peptide during the first 2 h of a 4-h mixed meal tolerance test at 1 year. Secondary outcomes included changes in glycated haemoglobin A(1c) (HbA(1c)) and insulin dose, and safety. Analysis included all randomised patients with known measurements. This trial is registered with ClinicalTrials.gov, number NCT00529399.145 patients were enrolled and treated with GAD-alum (n=48), GAD-alum plus alum (n=49), or alum (n=48). At 1 year, the 2-h AUC of C-peptide, adjusted for age, sex, and baseline C-peptide value, was 0·412 nmol/L (95% CI 0·349-0·478) in the GAD-alum group, 0·382 nmol/L (0·322-0·446) in the GAD-alum plus alum group, and 0·413 nmol/L (0·351-0·477) in the alum group. The ratio of the population mean of the adjusted geometric mean 2-h AUC of C-peptide was 0·998 (95% CI 0·779-1·22; p=0·98) for GAD-alum versus alum, and 0·926 (0·720-1·13; p=0·50) for GAD-alum plus alum versus alum. HbA(1c), insulin use, and the occurrence and severity of adverse events did not differ between groups.Antigen-based immunotherapy therapy with two or three doses of subcutaneous GAD-alum across 4-12 weeks does not alter the course of loss of insulin secretion during 1 year in patients with recently diagnosed type 1 diabetes. Although antigen-based therapy is a highly desirable treatment and is effective in animal models, translation to human autoimmune disease remains a challenge.US National Institutes of Health.

Abstract

Inflatable penile prostheses (IPPs) are a well-established and reliable treatment for medication refractory erectile dysfunction. The most serious complication with IPPs is infection, with the reported incidence after primary placement 1% to 3% and after revision surgery 8% to 18%.The aim of this report is to describe an infected decommissioned IPP reservoir with Actinomyces neuii with successful preservation of a functioning implant.After 9 years of successful use with an IPP (AMS 700 CX) for Peyronie's disease and organic erectile dysfunction, a 79-year-old man underwent replacement with an AMS 700 LGX. The decommissioned reservoir was kept in the right prevesical space, and the new reservoir was placed in the left prevesical space. Three months later, he presented with right inguinal pain and swelling.He was found to have an infected right reservoir with A. neuii, sparing his new IPP. After removal of the right reservoir, he had an uneventful recovery and has shown no evidence of infection in the new device.Revision surgery for IPPs carries a higher risk for implant infection. This is the first report of a genitourinary implant infection with A. neuii. Aggressive surgical and medical treatment may allow preservation of the functioning implant, despite gross infection of the decommissioned reservoir.

Abstract

Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.

Abstract

Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular 'glue' directly mediating T cell-DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). The critical factors which determined the extent of DC-T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC-T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC-T cell interactions at the IS.

Abstract

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.

Abstract

In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2-dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects.Persistence of Tregs was assessed by culturing sorted CD4(+)CD25(hi) natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4(+)CD25(-) T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis.Maintenance of FOXP3 expression in CD4(+)CD25(+) Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4(+) T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling.Aberrant IL-2R signaling in CD4(+) T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes.

Abstract

The composition of the ECM provides contextual cues to leukocytes in inflamed and healing tissues. One example of this is HA, where LMW-HA, generated during active inflammation, is a TLR ligand and an endogenous "danger signal," and HMW-HA, predominant in healing or intact tissues, functions in an inverse manner. Our data suggest that HMW-HA actively promotes immune tolerance by augmenting CD4+CD25+ T(Reg) function, and LMW-HA does not. Using a human iT(Reg) model, we demonstrate that HMW-HA but not LMW-HA provides a costimulatory signal through cross-linking CD44 which promotes Foxp3 expression, a critical signaling molecule associated with T(Reg). This effect, in part, may be mediated by a role for intact HMW-HA in IL-2 production, as T(Reg) are highly IL-2-dependent for their survival and function. We propose that HMW-HA contributes to the maintenance of immune homeostasis in uninjured tissue and effectively communicates an "all-clear" signal to down-regulate the adaptive immune system through T(Reg) after tissue matrix integrity has been restored.

Abstract

Work by our group and others has demonstrated a role for the extracellular matrix receptor CD44 and its ligand hyaluronan in CD4(+)CD25(+) regulatory T cell (Treg) function. Herein, we explore the mechanistic basis for this observation. Using mouse FoxP3/GFP(+) Treg, we find that CD44 costimulation promotes expression of FoxP3, in part through production of IL-2. This promotion of IL-2 production was resistant to cyclosporin A treatment, suggesting that CD44 costimulation may promote IL-2 production through bypassing FoxP3-mediated suppression of NFAT. CD44 costimulation increased production of IL-10 in a partially IL-2-dependent manner and also promoted cell surface TGF-beta expression. Consistent with these findings, Treg from CD44 knockout mice demonstrated impaired regulatory function ex vivo and depressed production of IL-10 and cell surface TGF-beta. These data reveal a novel role for CD44 cross-linking in the production of regulatory cytokines. Similar salutary effects on FoxP3 expression were observed upon costimulation with hyaluronan, the primary natural ligand for CD44. This effect is dependent upon CD44 cross-linking; while both high-molecular-weight hyaluronan (HA) and plate-bound anti-CD44 Ab promoted FoxP3 expression, neither low-molecular weight HA nor soluble anti-CD44 Ab did so. The implication is that intact high-molecular weight HA can cross-link CD44 only in those settings where it predominates over fragmentary LMW-HA, namely, in uninflamed tissue. We propose that intact but not fragmented extracellular is capable of cross-linking CD44 and thereby maintains immunologic tolerance in uninjured or healing tissue.

Abstract

Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic beta-cell function and homeostasis. We first examined beta-cells histologically and found that Myd88-/- mice have smaller islets in comparison to C57Bl/6 controls. Myd88-/- mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88-/-mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88-/- mice suffer enhanced beta-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on beta-cells primarily in the setting of injury.

Abstract

Hyaluronan is a glycosaminoglycan present in the extracellular matrix. When hyaluronan is degraded during infection and injury, low m.w. forms are generated whose interactions influence inflammation and angiogenesis. Intact high m.w. hyaluronan, conversely, conveys anti-inflammatory signals. We demonstrate that high m.w. hyaluronan enhances human CD4(+)CD25(+) regulatory T cell functional suppression of responder cell proliferation, whereas low m.w. hyaluronan does not. High m.w. hyaluronan also up-regulates the transcription factor FOXP3 on CD4(+)CD25(+) regulatory T cells. These effects are only seen with activated CD4(+)CD25(+) regulatory T cells and are associated with the expression of CD44 isomers that more highly bind high m.w. hyaluronan. At higher concentrations, high m.w. hyaluronan also has direct suppressive effects on T cells. We propose that the state of HA in the matrix environment provides contextual cues to CD4(+)CD25(+) regulatory T cells and T cells, thereby providing a link between the innate inflammatory network and the regulation of adaptive immune responses.

Abstract

CD1-restricted T cells have been shown to play a critical role in host defence, tumour surveillance, and maintenance of tolerance. However, immunologic outcomes resulting from activation of CD1d-restricted T cells can be either beneficial or deleterious. A major mechanism by which CD1d-restricted T cells are thought to exert immunoregulatory control is via effects on dendritic cell (DC) differentiation and migration. Important functional subsets of CD1d-restricted T cells are also known to exist and the potential implications for preferential subset activations are discussed.

Abstract

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Abstract

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus.

Abstract

A comparison of 25 hepatitis B virus (HBV) isolates for which complete genome sequences are available revealed two that occupied different positions in phylogenetic trees reconstructed from different open reading frames. Further analysis indicated that this incongruence was the result of recombination between viruses of different genomic and antigenic types. Both putative recombinants originated from geographic regions where multiple genotypes are known to cocirculate. A search of the sequence databases showed evidence of similar intergenotypic recombinants. These observations indicate that recombination between divergent strains may represent an important source of genetic variation in HBV.